TABLE 1.
Bacterial strains and plasmids
| Strain or plasmid | Properties | Source or reference |
|---|---|---|
| E. coli K-12 | ||
| DH5α | F−φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17 (rK− rM+) phoA supE44 λ− thi-1 gyrA96 relA1 | GIBCO-BRL |
| DH5α(λpir) | F−φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17 (rK− rM+) phoA supE44 thi-1 gyrA96 relA1 λpir | Laboratory stock |
| XL1-Blue | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac[F′ proAB lacIq ZΔM15 Tn10 (Tcr)] | Stratagene |
| Y. pestisa | ||
| KIM6 | pPCP1, pMT1 | R. R. Brubaker |
| KIM5-3001 | Smr; pCD1 (Lcr+), pPCP1, pMT1 | 30 |
| KIM5-3001.12 | Smr; pCD1 yscC [YscCΔ141–454]b, pPCP1, pMT1 | 51 |
| KIM5-3241 | pCD1 lcrV [LcrVΔ18–215], yopJ::MudI1734 (Kmr Lac+), pPCP1, pMT1 | 52 |
| KIM8-3241 | pCD1 lcrV [LcrVΔ18–215], yopJ::MudI1734 (Kmr Lac+), pMT1, pOS50; KIM5-3241 was cured of pPCP1 as described in Materials and Methods | This study |
| KIM8-3001.12 | Smr; pCD1 yscC [YscCΔ141–454], pMT1, pOS50; KIM5-3001.12, constructed previously (51), was cured of pPCP1 as described in Materials and Methods | This study |
| KIM8-3002 | Smr; pCD1 (Lcr+), pMT1 | 37 |
| KIM8-3002.7 | Smr; pCD1 ΔlcrG3[LcrGΔ6–86], pMT1 | This study |
| KIM8-3002.8 | Smr; pCD1 ΔlcrG2[LcrGΔ6–95] lcrV [LcrVΔ1–268], pMT1 | 42 |
| Plasmids | ||
| pProEX-1 | Apr; expression vector for creation of His6-fusion to N terminus of protein expressed from cloned open reading frame | GIBCO-BRL |
| pLD55 | Apr Tcr; suicide vector for allelic exchange | 32 |
| pBAD18-Kan | Kmr; ParaBAD cloning vector | 17 |
| pTrc99A | Apr; Ptrc cloning vector | Pharmacia |
| pOS50 | Tcr; competitive plasmid for pPCP1 | J. Goguen |
| Constructs | ||
| pES6-1 | HindIII-G of Y. pestis pCD1 cloned into pUC19 (lcrGVH yopBD′)c | 65 |
| pHT-V | PCR-amplified lcrV cloned in pProEX-1 in frame with leader sequence encoding 23 amino acids, including 6 His (lcrV) | This study |
| pHT-VN68 | Subcloned lcrV in pProEX-1 with a 29-residue His6 leader sequence fused at the EcoRV site in lcrV (′lcrV [HT-V Δ1–67 ≡ HT-VN68; same as HT-′V previously described (14)]) | This study |
| pHT-′V2 | EcoRV fragment of pES6-1 in pProEX-1 with the same 29-residue His6 leader sequence as in pHT-VN68 fused at the EcoRV site in lcrV (′lcrV [HT-VΔ1–67 ≡ HT-′V] lcrH yopB′ YopB1–87]) | 14 |
| pTrcV | BbvI/NcoI fragment of pES6-1 cloned into SmaI-cut pTrc99A (lcrV) | This study |
| pAraG18K | lcrG cloned into pBAD18-Kan (lcrG) | 41 |
| pAraV18K | lcrV cloned into pBAD18-kan (lcrV) | 41 |
| pAraGV18K | lcrGand lcrV cloned into pBAD18-Kan (lcrG lcrV) | 41 |
| pMNΔlcrG3 | ΔlcrG3 allele [lcrGΔ6–86] cloned into pLD55 | This study |
| pVC216 | PCR-amplified lcrV encoding amino acids 1–216 in pTrc99A (lcrV′ [LcrVΔ217–327]) | This study |
| pE15VN68 | YopE mRNA secretion signal (45 nt) fused to EcoRV site in lcrV; expression from yopE promoter (yopE′ ′lcrV [LcrVΔ1–67]) | This study |
a
All strains are Pgm− (73).
b
Numbers in brackets indicate residues deleted from gene product.
c
Descriptions in parentheses give relevant genes present on construct.